Microemulsion liquid chromatographic method for simultaneous separation and determination of six flavonoids of Apocynum venetum leaf extract

•A microemulsion liquid chromatography (MELC) method has been developed.•The microemulsion was used as mobile phase in the separation and determination of six flavonoids.•The method was investigated in detail.

A simple, cost-effective, and efficient method was developed for the rapid simultaneous separation and determination of six flavonoids (rutin, hyperoside, quercetin-3-O-sophoroside, isoquercitrin, astragalin and quercetin) of Apocynum venetum leaf extract by reversed phase high performance liquid chromatography using a microemulsion system mixture as the mobile phase. Separations were performed on the Zorbax Extend-C18 column with UV detection at 360 nm. The flow rate was 0.8 mL min−1. The optimized microemulsion mobile phase consisted of 2.5% (v/v) n-butanol, 1.2% (v/v) of Genapol X-080, 0.5% (v/v) ethyl acetate and 95.8% (w/v) of aqueous 20 mM phosphoric acid, pH adjusted to 6.0 with 0.3% triethylamine. Under the optimized conditions, the calibration curve for six flavonoids was linear in the range of 5–1000 μg mL−1 with the correlation coefficients greater than 0.9994. The intra-day and inter-day precision (RSD) were below 8.11% and the limits of detection (LOD) for the six flavonoids were 1.7–6.0 μg mL−1 (S/N = 3). The microemulsion liquid chromatography (MELC) method was successfully applied to separate and determine the six flavonoids of A. venetum leaf extract.